Photochemical regeneration of flavoenzymes - An Old Yellow Enzyme case-study.

Direct, NAD(P)H-independent regeneration of Old Yellow Enzymes represents an interesting approach for simplified reaction schemes for the stereoselective reduction of conjugated C=C-double bonds. Simply by illuminating the reaction mixtures with blue light in the presence of sacrificial electron donors enables to circumvent the costly and unstable nicotinamide cofactors and a corresponding regeneration system. In the present study, we characterise the parameters determining the efficiency of this approach and outline the current limitations. Particularly, the photolability of the flavin photocatalyst and the (flavin-containing) biocatalyst represent the major limitation en route to preparative application.

Relationship between radiosensitivity and Nrf2 target gene expression in human hematopoietic stem cells.

NFE2-related factor 2 (Nrf2), which belongs to the cap "n" collar family of basic region leucine zipper transcription factors, is a key protein in the coordinated transcriptional induction of expression of various antioxidant genes. The purpose of this study was to analyze the expression of Nrf2 target genes, such as heme oxygenase 1 (HO-1), ferritin heavy polypeptide 1 (FTH1), NAD(P)H dehydrogenase, quinone 1 (NQO1), glutamate-cysteine ligase catalytic subunit, glutamate-cysteine ligase modifier subunit, glutathione reductase (GSR) and thioredoxin reductase 1 (TXNRD1), after X irradiation of CD34(+) cells that were prepared from human placental/umbilical cord blood hematopoietic stem cells (HSCs). We evaluated the relationship between radiosensitivity and expression of Nrf2 target genes in HSCs. The number of colony-forming cells derived from 2-Gy-irradiated HSCs decreased to approximately 20% of the nonirradiated control. At the same time, the mRNA expression of HO-1, FTH1, NQO1, GSR and TXNRD1 was significantly increased after X irradiation. A statistically significant negative correlation was observed between the surviving fraction of HSCs and the intrinsic NQO1 mRNA expression, indicating that HSCs in which NQO1 mRNA levels are low may also be radioresistant. The present results suggest that the antioxidant system associated with Nrf2 is involved in the radiosensitivity of HSCs.

The function of chloroplastic NAD(P)H dehydrogenase in tobacco during chilling stress under low irradiance.

The function of chloroplastic NAD(P)H dehydrogenase (NDH) was examined by comparing a tobacco transformant (DeltandhB) in which the ndhB gene had been disrupted with its wild type, upon exposure to chilling temperature (4 degrees C) under low irradiance (100 micro mol m(-2) s(-1) PFD). During the chilling stress, the maximum photochemical efficiency of PSII (F(v)/F(m)) decreased markedly in both the wild type and DeltandhB. However, both F(v)/F(m) and P700(+), as well as the PSII-driven electron transport rate (ETR), in DeltandhB were lower than that in the wild type, implying that NDH-dependent cyclic electron flow around PSI functioned to protect the photosynthetic apparatus from chilling stress under low irradiance. Under the stress, non-photochemical quenching (NPQ), particularly the fast relaxing NPQ component (qf) and the de-epoxidized ratio of the xanthophyll cycle pigments, (A+Z)/(V+A+Z), were distinguishable in DeltandhB from those in the wild type. The lower NPQ in DeltandhB might be related to an inefficient proton gradient across thylakoid membranes (DeltapH) because of lacking an NDH-dependent cyclic electron flow around PSI at chilling temperature under low irradiance.

[Activity of NADP-dependent dehydrogenase of thymocytes and blood lymphocytes after irradiation of rabbits in vivo with long-wave laser radiation]. / Aktivnost' HADF-zavisimykh degidrogenaz timotsitov i limfotsitov krovi posle oblucheni krolikov in vivo dlinnovolnovym lazernym islucheniem.

To study the mechanisms of immunocompetent cells' response to long-wave laser radiation in male Chinchilla rabbits after in vivo emission (lambda = 632.8 nm; emissive power, 19 mW) for 15 min, lymphocytes and thymocytes were identified in the projection areas of the thymus and femur and the levels of NADP-dependent dehydrogenases were measured. The changes found in the levels of thymocyte NADP-dependent dehydrogenases while exposing the rabbits to radiation in the area of the femur, as well as the area-related differences in the modulating effects of laser radiation on the activity of the lymphocytic enzymes under study indicate the mediated cellular metabolic regulation which is likely to be chiefly affected by the autonomic nervous system in the first postradiation minutes.

NADPH-diaphorase distribution in the choroid after continuous illumination.

The role of nitric oxide (NO) in the regulation of the tone of the blood choroid vessels was investigated using the histochemical NADPH-diaphorase technique. Rats were continuously illuminated with white light, 12,000 lux, for 8 days and perfused after 1, 3, 10 and 20 days of total darkness. Cryostat sections were processed using the NADPH diaphorase histochemical method. An increase in reactive fibres was observed during days 1, 3 and 10, but after 20 days in darkness, reactivity was similar to that observed in control sections. These results indicate that NO plays a role in regulating the blood flow in the uveal vessels, and that there are changes in NO level dependent on the functional state of the retina.

[The effect of long-wave laser radiation on the NADP-dependent dehydrogenase activity of the blood lymphocytes in healthy persons and pulmonary tuberculosis patients]. / Vliianie dlinnovolnovogo lazernogo izlucheniia na aktivnost' NADF-zavisimykh degidrogenaz limfotsitov krovi zdorovykh liudei i bol'nykh tuberkulezom legkikh.

A study was made to elucidate the in vitro effects of long-wave laser irradiation on NAD(P)-dependent lymphocytic dehydrogenases in peripheral blood of healthy subjects and patients with fibrous-cavernous tuberculosis of the lungs. In addition to suppression of T-cell immunity and reinforcement of humoral factors, such patients demonstrated predominance of catabolic processes over anabolic, reduction of the substrate flow by Krebs cycle. The above phenomena explain different responses to long-wave laser irradiation (632 nm) in healthy subjects and tuberculous patients who are noticed to develop more active transfer of the lipid catabolism products to glycolysis.

Photoaffinity inhibition of rat liver NAD(P)H dehydrogenase by 3-(alpha-acetonyl-p-azidobenzyl)-4-hydroxycoumarin.

NAD(P)H dehydrogenase was purified in four steps from a homogenate of rat liver. The final step was affinity chromatography on Sepharose coupled to 3,3'-(m-hydroxybenzylidene)bis(4-hydroxycoumarin). The purified enzyme was inhibited competitively with respect to NADH by 3-(alpha-acetonyl-p-nitrobenzyl)-4-hydroxycoumarin (acenocoumarin) (Ki = 1.7 microM). The acenocoumarin was converted into an azide which was used to photoaffinity inhibit the enzyme. Following photolysis in the presence of the azide, the enzyme was inactivated in proportion to the concentration of azide present during irradiation. A maximum of 35-40% inhibition could be achieved by a single irradiation at 254 nm for 1.5 min. This inhibition was noncompetitive with respect to NADH. The inactivation was shown to be specific as acenocoumarin afforded complete protection against inactivation, irradiation was required to achieve inactivation, and the enzyme was unaffected by irradiation alone.

[Biological oxidation in cells exposed to microwaves in the millimeter range]. / Biologicheskoe okislenie v kletke pri deistvii radiovoln millimetrovogo diapazona.

In the cells of RH, SPEV and HEp-2 lines irradiated with 6.5 mm radiowaves of 1 mW/cm2 flux density the following phenomena were established: activation of succinate dehydrogenase and ATPase; reduction of cytochrome oxidase, NAD- and NADP-diaphorase, acid and alkaline phosphatase activities; repression of 3H-thymidine incorporation in DNA and of 3H-uridine incorporation in RNA; violation of ultrastructure; suppression of cellular proliferation; decrease of mitotic activity; occurrence of pathological forms of mitosis.